bmc genetics biomed central...nias are hernia inguinalis and hernia scrotalis which occur within the...

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Open AcceResearch articleGenome-wide linkage analysis of inguinal hernia in pigs using affected sib pairsEli Grindflek*1,2, Maren Moe1,3, Helge Taubert4, Henner Simianer4, Sigbjørn Lien2,3 and Thomas Moen2,5
Address: 1The Norwegian Pig Breeders Association (NORSVIN), Hamar, Norway, 2Centre for Integrative Genetics, Norwegian University of Life Sciences, Aas, Norway, 3Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, Aas, Norway, 4Institute of Animal Breeding and Genetics, Georg-August University of Goettingen, Goettingen, Germany and 5AKVAFORSK, Aas, Norway

Email: Eli Grindflek* - [email protected]; Maren Moe - [email protected]; Helge Taubert - [email protected]; Henner Simianer - [email protected]; Sigbjørn Lien - [email protected]; Thomas Moen - [email protected]

* Corresponding author

AbstractBackground: Inguinal and scrotal hernias are of great concern to pig producers, and lead to pooranimal welfare and severe economic loss. Selection against these conditions is highly preferable, butat this time no gene, Quantitative Trait Loci (QTL), or mode of inheritance has been identified inpigs or in any other species. Therefore, a complete genome scan was performed in order to identifygenomic regions affecting inguinal and scrotal hernias in pigs. Records from seedstock breedingfarms were collected. No clinical examinations were executed on the pigs and there was thereforeno distinction between inguinal and scrotal hernias. The genome scan utilised affected sib pairs(ASP), and the data was analysed using both an ASP test based on Non-parametric Linkage (NPL)analysis, and a Transmission Disequilibrium Test (TDT).

Results: Significant QTLs (p < 0.01) were detected on 8 out of 19 porcine chromosomes. Themost promising QTLs, however, were detected in SSC1, SSC2, SSC5, SSC6, SSC15, SSC17 andSSCX; all of these regions showed either statistical significance with both statistical methods, orconvincing significance with one of the methods. Haplotypes from these suggestive QTL regionswere constructed and analysed with TDT. Of these, six different haplotypes were found to bedifferently transmitted (p < 0.01) to healthy and affected pigs. The most interesting result was onehaplotype on SSC5 that was found to be transmitted to hernia pigs with four times higher frequencythan to healthy pigs (p < 0.00005).

Conclusion: For the first time in any species, a genome scan has revealed suggestive QTLs foringuinal and scrotal hernias. While this study permitted the detection of chromosomal regions only,it is interesting to note that several promising candidate genes, including INSL3, MIS, and CGRP,are located within the highly significant QTL regions. Further studies are required in order tonarrow down the suggestive QTL regions, investigate the candidate genes, and to confirm thesuggestive QTLs in other populations. The haplotype associated with inguinal and scrotal herniasmay help in achieving selection against the disorder.

Published: 03 May 2006

BMC Genetics 2006, 7:25 doi:10.1186/1471-2156-7-25

Received: 02 February 2006Accepted: 03 May 2006

This article is available from: http://www.biomedcentral.com/1471-2156/7/25

© 2006 Grindflek et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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BMC Genetics 2006, 7:25 http://www.biomedcentral.com/1471-2156/7/25

BackgroundThe occurrence of hernias is a significant problem facingpig producers, and leads to poor animal welfare andsevere economic loss. The most commonly occurring her-nias are hernia inguinalis and hernia scrotalis which occurwithin the pig population at frequencies from 1.7% to6.7% [1]. By definition, an inguinal hernia describes a sit-uation where hernial contents are present in the inguinalcanal, whereas scrotal hernia refers to a situation wherehernial contents are present in the scrotum. In these con-ditions, most frequently the distal jejunum and ileumpass through the vaginal ring and enter the inguinal canal.Small colon and omentum can also herniate but thisoccurs less frequently. Inguinal and scrotal hernias are fur-ther subdivided, in human medicine, as indirect or direct.An indirect hernia refers to the passage of intestinal loopsthrough the vaginal ring into the vaginal tunic. While adirect hernia refers to the passage of intestinal loopsthrough a fascial defect near the vaginal ring, resulting ina situation where intestinal loops pass through theinguinal canal but are not covered by the vaginal tunic.The inguinal canal is a short passage running through theinferior part of the interior abdominal wall. Under nor-mal conditions its function is to allow the testes, whichdevelop in the lumbar region of the abdomen, to migrateto the scrotum. The testes descend from the anteriorabdominal wall through the processus vaginalis via a pro-pulsive force generated by muscles derived from thegubernaculums, a ligament linking the testes and scro-tum. Development of inguinal and scrotal hernias usuallyresults from failed obliteration of the processus vaginalisafter descent of the testis [2], or from failed involution atthe internal inguinal ring [3]. Complication may alsooccur, in the form of intestinal obstruction or strangula-tion [4]. Furthermore, in many cases the undescended tes-tes are associated with a patent processus vaginalis becausethe processus vaginalis does not obliterate unless the testisreach the scrotum [5]. This particular form of inguinal andscrotal hernias is correlated with the occurrence of cryp-torchism [1], and is a concern for the pig breeding indus-try as well.

Several studies have shown that genetic factors areinvolved in the development of inguinal and scrotal her-nias [6,7], however the mode of inheritance has not yetbeen clarified. Estimated h2 for these types of hernia rangefrom 0.20 [8] to 0.86 [9]. Several studies have investigatedgenes involved in the control of testicular descent, obliter-ation of processus vaginalis and in the closing of theinguinal ring; by association, these genes may also beinvolved in the occurrence of inguinal and scrotal hernias.Specifically, Insulin-like receptor 3 (INSL3), Müllerianinhibiting substance (MIS), and relaxin are all involved ingubernacular growth [10], while the calcitonin gene-related peptide (CGRP) released from the genitofemoral

nerve, may be responsible for failed fusion and disappear-ance of processus vaginalis [11,12].

The aim of this study was to identify genomic regionsaffecting the frequency of inguinal and scrotal hernias inpigs. Records from seedstock breeding farms were used inthis study. No clinical examinations were executed on thepigs and therefore, there was no distinction betweeninguinal and scrotal hernias. Examination of these regionsmay reveal causative genes responsible for the trait, whichwould be valuable information for use in Marker-AssistedSelection (MAS) schemes directed at decreasing herniaprevalence. Previously, parametric linkage analysisapplied to large pedigrees containing many affected indi-viduals has helped in the identification of genes with highpenetrance [13]. However, for those diseases lacking aclear Mendelian inheritance pattern, or caused by severalgenes of low to moderate penetrance, nonparametricanalysis can be a more robust and successful alternative.Crucially, linkage analysis requires correct assumptions tobe made regarding the inheritance model and allele fre-quencies of the susceptibility alleles, and incorrect modelspecification will result in a loss of power and a bias in theestimation of recombination fraction [14]. Nonparamet-ric methods, by comparison, require no such assumptionsto be made, and we consider this to be better suitedapproach for our analysis of small sets of affected rela-tives. The Transmission/Disequilibrium Test (TDT) [15]was used to investigate whether the identified hernia-linked markers were associated with the trait on a popula-tion level. In the present article, we describe the results ofthe first published genome-wide scan of inguinal andscrotal hernias in pigs, as well as one of the first genomescans in livestock populations to use the affected sib-pairdesign.

ResultsLinkage analysisThe linkage analysis was performed using an affected sibpair (ASP) test. Figure 1 and 2 shows the profiles of themultipoint NPL scores and the respective informationcontent (Info) for each chromosome. The average infor-mation content was 0.52, which is a similar value to thosefound in previous studies in swine [16] and humans [17].Two chromosomal regions had linkage score exceedingthe nominal significance level of p < 0.001. The most sig-nificant regions were found on SSC2, with a multipointNPL score of 3.16 at marker SW834, and on SSC15 whichshowed an NPL score of 3.47 at marker SW919. Fourmarkers on SSC2 reached a significance level of p < 0.01(NPL score larger than 2.33); these four markers spanneda chromosomal region of 40 cM. In addition, the linkagescore exceeded the nominal significance level of p < 0.01for six chromosomes, with highest maximum multipointNPL scores of 2.35 at marker S0301 on SSC4, 2.45 at




Linkage results of the total genome scan for inguinal and scrotal hernias, showing sus scrofa chromosomes (SSC) 1 to 12Figure 1Linkage results of the total genome scan for inguinal and scrotal hernias, showing sus scrofa chromosomes (SSC) 1 to 12. The bold line shows multipoint NPL score (scale on left) while the dotted line shows the information content (scale on right) for each chromosome. The threshold values P = 0.05 and P = 0.01 are shown as horizontal lines at NPL = 1.64 and NPL = 2.33, respectively. Marker positions and names are shown on the X axis.

SSC4

S0001

S0301

S0227

A-FABP

SW839

SW445

S0214

S0073

20 40 60 80 100 120

0

0,5

1

1,5

2

2,5

3

3,5

4

NPL

0

0,2

0,4

0,6

0,8

1

Info

SSC2

SWR308

SW1844

SWR2157

SW2134

SW834

SW1686

SW1650

SWR2516

20 40 60 80 100 120

0

0,5

1

1,5

2

2,5

3

3,5

4

NPL

0

0,2

0,4

0,6

0,8

1

Info

SSC5

SW1954

SWR1526

SW963

SW1071

SW1482

SW491

SW413

12010080604020

0

0,5

1

1,5

2

2,5

3

3,5

4

NPL

0

0,2

0,4

0,6

0,8

1

Info

SSC8

SW2410

SW205

SW1679

SW2160

S0144

SW790

SW1980

20 40 60 80 100 120

0

0,5

1

1,5

2

2,5

3

3,5

4

NPL

0

0,2

0,4

0,6

0,8

1

Info

SSC9

SW2116

SW989

SW2495

SW2074

SW827

SW911

SW983

12010080604020

0

0,5

1

1,5

2

2,5

3

3,5

4

NPL

0

0,2

0,4

0,6

0,8

1

Info

SSC10

SW830

SW767

S0351

SWR1849

SW2043

SW1626

SWR67

20 40 80 100 12060

0

0,5

1

1,5

2

2,5

3

3,5

4

NPL

0

0,2

0,4

0,6

0,8

1

Info

SSC3

SW2532

SW142

SW2141

SWR978

SW487

SW72

S0213

12010080604020

0

0,5

1

1,5

2

2,5

3

3,5

4

NPL

0

0,2

0,4

0,6

0,8

1

Info

SSC11

SW1684

SW1452

SW1377

SW903

SW66

S0392

S0391

12010080604020

0

0,5

1

1,5

2

2,5

3

3,5

4

NPL

0

0,2

0,4

0,6

0,8

1

Info SSC12

SW2490

SW2494

SW957

SW1553

SW1956

S0147

SW2180

20 40 60 80 100 120

0

0,5

1

1,5

2

2,5

3

3,5

4

NPL

0

0,2

0,4

0,6

0,8

1

Info

SSC1

SW1515

SW1332

S0008

SW1430

S0331

SW1311

SW1512

20 40 60 80 100 120

0

0,5

1

1,5

2

2,5

3

3,5

4

NPL

0

0,2

0,4

0,6

0,8

1

Info

SSC6

SW2406

SW1057

S0087

SW1355

SW1823

S0003

SW322

SW2419

20 40 60 80 100 120

0

0,5

1

1,5

2

2,5

3

3,5

4

NPL

0

0,2

0,4

0,6

0,8

1

Info

SSC7

SW1303

S0212

SW632

SW352

SW175

S0102

TNFB

S0064

S0025

14012010080604020

0

0,5

1

1,5

2

2,5

3

3,5

4

NPL

0

0,2

0,4

0,6

0,8

1

Info



Linkage results of the total genome scan for inguinal and scrotal hernias, showing sus scrofa chromosomes (SSC) 13 to XFigure 2Linkage results of the total genome scan for inguinal and scrotal hernias, showing sus scrofa chromosomes (SSC) 13 to X. The bold line shows multipoint NPL score (scale on left) while the dotted line shows the information content (scale on right) for each chromosome. The threshold values P = 0.05 and P = 0.01 are shown as horizontal lines at NPL = 1.64 and NPL = 2.33, respectively. Marker positions and names are shown on the X axis.

SSC13

S0282

SW1378

SWR1008

S0223

S0075

SW1056

S0289

S0291

20 40 60 80 100 120

0

0,5

1

1,5

2

2,5

3

3,5

4

NPL

0

0,2

0,4

0,6

0,8

1

Info SSC14

SW619

SW2038

SW510

S0162

SW2519

SW886

SW1081

SW1557

SWC27

20 40 60 80 100 120

0

0,5

1

1,5

2

2,5

3

3,5

4

NPL

0

0,2

0,4

0,6

0,8

1

Info

SSC15

SW1416

SW919

SW1118

SW1989

SW1316

SW1983

SWR2121

20 40 60 80 100 120

0

0,5

1

1,5

2

2,5

3

3,5

4

NPL

0

0,2

0,4

0,6

0,8

1

Info

SSC16

S0105

SW2517

SW977

SW81

SW1035

SW419

SW1305

12010080604020

0

0,5

1

1,5

2

2,5

3

3,5

4

NPL

0

0,2

0,4

0,6

0,8

1

Info

SSC17

SW1891

SWR1120

SW2441

S0292

S0359

S0332

SW2427

20 40 60 80 100 120

0

0,5

1

1,5

2

2,5

3

3,5

4

NPL

0

0,2

0,4

0,6

0,8

1

InfoSSC18

SW1808

SW1984

SW1682

20 40 60 80 100 120

0

0,5

1

1,5

2

2,5

3

3,5

4

NPL

0

0,2

0,4

0,6

0,8

1

Info

SSCX

SW2059

SW2588

SW1943

SW259

SW1522

SW1903

SW1325

12010080604020

0

0,5

1

1,5

2

2,5

3

3,5

4

NPL

0

0,2

0,4

0,6

0,8

1

Info


marker SW963 on SSC5, 2.92 at marker S0075 on SSC13,2.47 at marker SW1891 on SSC17, 2.80 at markerSW1808 on SSC18, and 2.74 at marker SW259 on SSCX.

Finally, the nominal significance level of p < 0.05 wasreached on four chromosomal regions with highest maxi-mum multipoint NPL scores of 2.06 at marker S0331 on

Table 1: Comparison of results of affected sib pair test (ASP) and transmission disequilibrium test (TDT). The chromosome positions and single-point levels of significance are shown for all significant markers in the genome scan. The significant p-values (1%) are in bold.

Chromosome Marker Position (cM) P-value, ASP P-value, TDT

SSC1 SW1515 0 0.05 0.07SW1332 20 0.05 0.005S0331 60 0.05 0.05

SSC2 SW1686 45 0.01 0.23SW834 65 0.001 0.86SW2134 75 0.002 0.17SWR2157 85 0.001 0.90SWR308 125 0.01 0.06

SSC4 S0227 0 0.05 0.49S0301 20 0.01 0.53S0214 70 0.05 0.61

SSC5 SW963 * 80 0.01 0.005SSC6 SW1355 * 60 0.11 0.0002

S0003 80 0.05 0.18SSC7 TNFB 55 0.05 0.10

S0212 135 0.05 0.05SSC12 SW2180 105 0.05 0.18SSC13 S0223 60 0.01 0.40

S0075 65 0.01 0.93SSC15 SW919 20 0.001 0.02

SW1118 35 0.05 0.01SSC17 SW1891 0 0.01 0.01

SWR1120 10 0.05 0.74SW2441 25 0.05 0.11S0332 70 0.05 0.01

SSC18 SW1984 30 0.05 0.17SW1682 45 0.01 0.72

SSCX SW1522 50 0.006 0.28SW259 70 0.003 0.53

* Markers with chromosomewise significance of p < 0.01 from the TDT analyses

Table 3: The significant haplotypes from the most significant QTL regions were analysed by TDT. The counts are the number of times that an allele was transmitted/not transmitted from a heterozygous parent carrying that allele to a hernia affected/not affected offspring. Nominal significance levels were used.

Chromosome Markers in the haplotype

Affected Non-affected TDT P-value

Transmit. Not transmit. Transmit. Not transmit.

SSC5 SW963-SWR1526

41 16 10 24 16.7 0.00004

SSC6 SW1355-SW1823-S0003

30 13 6 13 9.3 0.002

SSC15 SW919-SW1118

56 36 16 28 7.5 0.006

5 15 8 3 7.3 0.007SSC17 _ 1 SW1891-

SWR11205 15 8 3 7.3 0.007

SSC17 _ 2 S0359-S0332 58 26 21 22 8.6 0.003



SSC1, 1.68 at marker S0003 on SSC6, 2.20 at markerTNFB on SSC7, and 2.06 at marker SW2180 on SSC12.Results with significance level above 5% are presented inTable 1.

TDT analysisTDT analysis was applied to all loci in the data set. The sig-nificant loci are presented in Table 1. At the TDT-signifi-cant loci, TDT was also performed on the allele level, toidentify individual hernia-associated alleles (Table 2).Markers displaying significance at a nominal value of p <

Table 2: Counts used as input for TDT, with results from single-allele TDT. The counts are the number of times that an allele was transmitted/not transmitted from a heterozygous parent carrying that allele to a hernia affected/not affected offspring. Nominal significance levels were used.

Chromosome

Marker Allele Affected Resistant TDT P-value

Transmit. Not transmit. Transmit. Not transmit.

SSC1 S0331 1 49 14 27 20 7.13 0.0072 10 25 11 13 2.86 0.093 13 25 10 15 0.78 0.384 0 8 2 2 5.33 0.025 4 4 3 3 0.00 1.00

SW1332 1 22 35 7 23 0.10 0.752 54 24 25 16 3.71 0.053 1 14 5 2 11.64 0.00064 1 5 4 0 6.40 0.011

SSC5 SW963 1 53 25 13 25 13.79 0.00022 8 10 3 7 0.14 0.703 27 37 17 10 3.18 0.074 27 35 19 14 1.78 0.185 8 10 7 1 2.46 0.126 1 5 1 3 0.40 0.537 0 2 1 1 1.00 0.32

SSC6 SW1355 1 48 70 48 19 14.06 0.00022 16 14 1 17 6.75 0.0093 41 35 18 21 0.70 0.404 38 24 12 22 6.00 0.01

SSC7 S0212 1 9 11 8 7 0.26 0.612 52 29 17 20 5.73 0.013 1 12 0 7 0.80 0.374 18 21 15 7 1.98 0.165 25 33 18 12 2.23 0.147 5 4 1 6 2.25 0.13

SSC15 SW1118 1 40 21 15 24 7.84 0.0052 11 16 8 7 0.86 0.353 9 17 10 6 3.43 0.064 2 9 3 3 2.88 0.095 7 6 6 2 0.43 0.51

SW919 1 52 35 13 27 7.57 0.0062 18 33 15 18 1.71 0.193 37 35 32 7 4.77 0.034 32 34 18 16 0.16 0.695 8 10 0 10 2.29 0.136 1 1 1 1 0.00 1.00

SSC17 S0332 1 24 31 14 12 1.00 0.322 1 21 0 10 3.13 0.084 30 42 15 34 0.40 0.525 7 20 9 4 8.10 0.004

SW1891 1 60 39 32 29 2.03 0.152 27 41 28 7 11.89 0.00053 19 24 9 20 0.50 0.484 25 27 9 22 1.46 0.22



0.05 were found on SSC1, SSC5, SSC6, SSC7, SSC15, andSSC17. A chromosome-wide significance of p < 0.01 wasfound for the two markers; SW963 on SSC5 and SW1355on SSC6. All TDT-significant markers were also significantin the ASP test, with the exception of SW1355 on chromo-some 6, which was also the most significant marker foundin the TDT.

The loci showing significance for both ASP and TDT, andthe regions showing high significance (p < 0.005) withonly ASP or TDT, were considered to be the most interest-ing for further analyses. Haplotypes from these regionswere constructed and TDT analysis was used to identifythose haplotypes most strongly associated with hernia. Sixdifferent haplotypes were found to be differently trans-mitted (p < 0.01). One haplotype on SSC5 was transmit-ted four times more frequently to pigs with hernias thanto healthy pigs (p < 0.00005). Other haplotypes, showingan increased frequency of transmission to affected pigs (p< 0.01) were found on SSC6, SSC15 and SSC17. Table 3shows the markers included in the haplotypes, thenumber of affected/non-affected animals with the trans-mitted/non-transmitted haplotype, and the p-values. Dis-tributions belonging only to the significant haplotypes areshown.

DiscussionOur genomewide scan for QTLs affecting incidence of her-nia revealed 9 genomic regions on 8 chromosomes usingASP test (p < 0.01), and 6 genomic regions on 6 chromo-somes using TDT (p < 0.01). Two of these regions, onSSC5 and SSC17, were significant (p < 0.01) when ana-lysed by both statistical methods (Table 1). Five regionslocated on SSC1, SSC2, SSC6, SSC15 and SSCX werefound to be highly significant (p < 0.005) from either theASP test or the TDT. We consider these seven regions to bethose most likely involved in genetic susceptibility to her-nia. Two different statistical methods were used (ASP andTDT) in order to take advantage of their complementaryqualities in detecting genomic regions affecting defect/dis-ease traits. The ASP method tests for markers linked togenes affecting deformity status, while the TDT methodtests for markers that are both linked to these genes andassociated with a particular trait on a population level.Essentially, the TDT is a fine-mapping method requiringlinkage disequilibrium (LD) between causative polymor-phisms and the nearest markers. In this study however,the TDT has been used on families with more than one(affected or unaffected) offspring per parental pair; undersuch conditions the TDT is, to some extent, a test for link-age, although the power of the test still increases greatlywith the amount of LD [18]. Therefore, for our applica-tion, the ASP and TDT are seen as two different tests forgenes linked to the trait, possessing complementarystrengths and weaknesses. In particular, the ASP is

expected to be the more powerful test when the causativepolymorphism is linked to, but not in LD with, the near-est marker, whereas the TDT would have greater power ifsome amount of LD were present. Other factors, such aspenetrance and allele frequencies in causative polymor-phisms and markers, may also lead to differences inpower between the two tests [19]. Previous studies haveshown that the two methods may give different results.For example, the TDT detected a very strong associationbetween diabetes mellitus and a marker in the 5' region ofthe insulin gene [20,21], although this marker had earlierbeen shown to have no association in an ASP-based testfor linkage [22,23]. In our study several genomic regionsare highly significant for the ASP test but not for TDT(Table 1). As the ASP test can detect linkage over long dis-tances, these results may be explained by markers beinglinked to, but not in close LD with, the causative polymor-phisms. The negative TDT results should therefore not beinterpreted as a negation of the findings from the ASP test,but rather as indications that the markers are not locatedin sufficient proximity to the causative polymorphisms. Infact, with the marker densities used in this study, ASP-pos-itive but TDT-negative markers are expected. However,one marker that was strongly positive for the TDT test(SW1355 on SSC6, p < 0.0002), was negative for the ASPtest. This may be explained by the fact that in contrast tothe ASP, the TDT method can detect association with highpower when homozygotes for a susceptibility allele haveas little as 2 to 4-fold greater disease risk than homozy-gotes for the normal or wild-type allele, while parameterssuch as penetrance and allele frequencies may also favourthe TDT rather than the ASP test [19].

The causative mutations we are seeking do not occur inisolation, but with a number of different mutationswithin the region. Linkage disequilibrium (LD) deterio-rates over successive generation because of recombina-tion, but the mutation will remain in LD with closelylinked markers. It is therefore, possible to define a riskmodifying haplotype in a region of the genome surround-ing the disease mutation. Once multiple single-pointassociations within a region have been identified, haplo-type based tests for disease associations may be per-formed. In this investigation, haplotypes consisting ofmarker alleles in the putative QTL regions were investi-gated using TDT analysis. Several haplotypes occurredwith a higher frequency in the affected pigs compared tothe unaffected pigs, indicating that these haplotypes areassociated with the occurrence of inguinal and/or scrotalhernias in pigs.

While most porcine genes are still not mapped, the deter-mination of homologous synteny blocks between humanand porcine chromosomes allows us to propose somecandidate genes for inguinal and scrotal hernias using



comparative genomics [24-27]. The comparative regionsand possible candidate genes within these regions areshown in Table 4. As mentioned previously, inguinal andscrotal hernias may arise from a failure to achieve com-plete closure of the processus vaginalis, and a failure ofinvolution at the internal inguinal ring [3]. In addition,the genitofemoral nerve, the caudal ligament gubernacu-lum, and controllers of testicular descent may control sub-sequent closure of the processus vaginalis. Finally,regulation of collagen metabolism has been shown toplay an important role in the development of inguinaland scrotal hernias [28,29]. Using this information as aguide it is possible to anticipate that certain candidategenes may be located in some of the putative QTL regions.Three interesting candidate genes (INSL3, CGRP, MIS)have been mapped to homologous regions in the putativeQTL on SSC2 [30-32]. From studies using transgenicknockout mice [33] and humans [34], INSL3 is believedto be the primary testicular hormone inducing gubernac-ular development, and has been associated with occur-rences of inguinal hernia in female mice [7]. Nosignificant associations were, however, observed betweentwo polymorphisms in the INSL3 gene and inguinal her-nia in pigs [35]. The CGRP is active in the induction ofprocessus vaginalis fusion, a process involving substantialtissue remodelling and the characteristic transformationof processus vaginalis epithelium [6,12]. Finally, MIS hasbeen shown to be involved in the swelling reaction ofgubernaculum occurring during the first phase of testicu-lar descent [12,36]. Moreover, MIS together with testoster-one and INSL3, controls sex differentiation-likedevelopment of internal and external reproductive organsand the acquisition of male secondary sex characteristics[37]. Additional candidate genes were found in putativeQTL regions using the network browser option in Pub-

Gene [38]. Estrogen receptor 1 (ESR1) and collagentypeIXα (COL9A1) are both candidate genes located tohuman (HAS) 6q25 region, which is comparative to theputative QTL on SSC1. Coveney et al. [39] demonstratedthat estrogen has profound effects on development of theinternal genitalia in male marsupials, preventing inguinalclosure and interfering with testicular descent, and ESR1knockout mice lack gubernaculum development in latestages of maturity [40]. Pathological changes in collagenare also involved in development of a hernia [41]. It istherefore interesting to notice that in cartilage, type IX col-lagen (COL9A1) molecules are covalently crosslinked totype II collagen molecules, which represents approxi-mately 85% of the collagen contained in hyaline cartilage3 [42]. Interestingly, the gene (COL2A1) coding for colla-gen type II has been mapped to a region in HSA12 whichis comparative to the putative QTL region on SSC5. Can-didate genes were also seen in the QTL regions on SSC6(INSL5) and SSC7 (CYP19A1). No obvious candidategenes were seen in the genomic regions of SSC7q24-26,SSC15 and SSC17 or in the comparative genomic regionsin human. The current comparative maps, however, donot allow precise prediction of the likely map position.

To our knowledge, this is the first report of a wholegenome scan for markers associated with inguinal andscrotal hernias, and one of the first genetic studies usingASP in livestock. The results reported in this study willencourage us to perform follow-up studies using finema-pping approaches in an extended genetic material. Ourgoal is to discover the causal mutations underlyinginguinal and/or scrotal hernias, and to look for sharedancestral haplotypes among pigs with these types of her-nias. Ultimately we wish to understand the epistatic inter-actions between inguinal/scrotal hernia genes, and the

Table 4: Putative candidate genes in candidate QTL regions for inguinal and scrotal hernias. The most promising regions on SSC1, SSC2, SSC5, SSC6, SSC7, SSC15 and SSC17 are shown, as well as the homologous regions in human.

QTL region1 Homologous regions in human2 Putative candidate genes

SSC1 p25 – q14 HSA6 q25 Collagen typeIXα (COL9A1), Estrogen receptor 1 (ESR1)

HSA18 p11.2 – p11.32SSC2 p14 – q23 HSA5 q12 – q35

HSA11 q13 – p15 Calcitonin gene-related peptide (CGRP)HSA19 p13.1 – p13.3 Insulin like hormone 3 (INSL3), Mullerian-

inhibiting substance (MIS)SSC5 q11 – q22 HSA12 q11.2 – p13 Collagen type II (COL2A1)SSC6 q27 – q32 HSA1 p22 – p36.3 Insulin-like hormone 5 (INSL5)

HSA18 q12 – p11.32SSC7 q11 – q15 HSA15 q12 – q26 Cytochrome P450, family19A1 (CYP19A1)SSC7 q24 – q26 HSA14 q11.2 – q32SSC15 HSA2 q11.2 – q21SSC17 p11 and p21 HSA5 and HSA20

1Based on the cytogenetic maps we suggest where the microsatellite markers are physically mapped to the porcine genome [48,49]2This is seen from comparative mapping between pig and human [24,25,26,27,50]



interactions between genes and their environment, andtheir impact on a variety of relevant cofactors associatedwith this defect. We believe the data contained in thepresent study represents an important and useful basis tofurther search for candidate genes causing inguinal andscrotal hernias in pigs. The outcome from these inquiriesmay also be of relevance to human hernia conditions.

ConclusionFor the first time in any species, a genome scan hasrevealed suggestive QTLs for inguinal and scrotal hernias.Significant QTLs were detected in 8 out of 19 porcinechromosomes (p < 0.01). However, the most promisingQTLs were found on SSC1, SSC2, SSC5, SSC6, SSC15,SSC17 and SSCX. These regions were found to be signifi-cant when using both statistical methods (ASP and TDT),or with a convincing high significance for either of themethods. Haplotypes from the suggestive QTL regionswere constructed and used in TDT analysis. Six differenthaplotypes were found to be differently transmitted whencontrasting hernia pigs with healthy pigs (p < 0.01). Onehaplotype on SSC5 was transmitted four times more fre-quently to hernia pigs than to healthy pigs (p < 0.00005).Only chromosomal regions could be detected in thisstudy, but it is noteworthy that several promising candi-date genes, including INSL3, MIS, and CGRP, are alllocated in a highly significant QTL region. Further studiesmust be performed to narrow down the suggestive QTLregions, investigate the candidate genes, and finally toconfirm the suggestive QTLs in other populations.

MethodsAnimals and phenotypic recordsDue to a generally low incidence of hernia inguinalis or her-nia scrotalis, it was necessary to utilise a collection systeminvolving all the breeding farms in Norsvin (Hamar, Nor-way) in order to access a sufficient number of affected sibpairs (ASP). The power calculations and minimumnumber of affected sib pairs required for this study wasevaluated based on a method suggested by Simianer andStricker [43]. Blood samples were collected from a total of100 full- and half-sib Norwegian Landrace piglets display-ing inguinal and/or scrotal hernia, along with blood from51 phenotypically unaffected full- and half-sibs. To sup-plement these samples, DNA from 27 fullsib Danish Lan-drace piglets affected with inguinal and/or scrotal hernias,and 16 unaffected siblings, was provided from DanishSlaughterhouses (Denmark). In total there were 194 sam-ples, distributed on 52 litters containing 103 affected sibpairs, with each litter containing 2 to 5 affected piglets.Two affected siblings from one litters gives 1 ASP, threeaffected siblings from one litters gives 3 ASP, and so on. Inaddition to affected animals and 1 or 2 unaffected sib-lings, we obtained blood specimens from all availableparents (31 sires and 46 dams). In several cases these were

parent to more than one affected litter. Samples wereobtained from 35 pig breeding farms altogether, and sev-eral sires were used on different farms.

When a farmer reported an affected animal, diagnosticprocedures were performed by a breeding consultant fromNorsvin. Without clinical examination, it is impossible todistinguish direct hernia from indirect hernia, or inguinalhernia from scrotal hernia. No clinical examinations wereexecuted, so the diagnosis of hernia includes bothinguinal and scrotal hernias, as well as both indirect anddirect hernias. In human and horses, inguinal and scrotalhernias are almost exclusively of the indirect type [44],therefore we expect most of the hernias to be of the indi-rect type in pigs also.

GenotypingGenomic DNA was extracted from a total of 282 pigs (par-ents and offspring). 130 microsatellite markers from theautosomal chromosomes and 7 microsatellites from theX/Y chromosome were selected from the USMARCGenome Database (2000), based on position, ease ofscoring and number of alleles [45]. The markers wereamplified using PCR; the reaction volume was 10 µl, con-taining 50 ng swine genomic DNA template, 10 × PCRbuffer, 15 mM MgCl2, 2 mM dNTP, 0.01 µm of eachprimer and 5U Gold Taq polymerase (Applied Biosys-tems, Foster City, CA, USA). The PCR program includedan initial denaturation step of 10 sec at 95°C, followed by40 cycles of 30 sec at 94°C, and 30 sec at 55°C-62°C, witha final extension step of 5 min at 73°C. Fragment lengthswere determined either by 1) electrophoresing PCR prod-uct through a 6% denaturating polyacrylamide gel in anABI-377 DNA sequencer (ABI, Perkin Elmer, Foster City,CA, USA), where each forward primer was 5'-labeled withone of three fluorophores (TET, HEX or 6-FAM), or 2)with capillary electrophoresis on an ABI 3730 DNAsequencer (ABI, Perkin Elmer, Foster City, CA, USA), eachforward primer 5'-labeled with one of four fluorophores(NED, PET, VIC or 6-FAM). Genescan and Genotyper ver-sion 3.7 software or GeneMapper 3.0 (ABI, Perkin Elmer,Foster City, CA, USA) was used for genotype scoring. Acomputer program was written in house to control forMendelian inconsistencies in the marker data.

Statistical analysisMarker mapThe family material used in the study was not suitable forconstructing genetic maps. Therefore the order of the 137microsatellites chosen for the genome scan, and thegenetic distances between them, were taken from theUSMARC Genome Database (2000). The markers wereevenly distributed across the 18 autosomal chromosomesand the X-chromosome, including one marker in thepseudoautosomal region. The total length of the map



spanned by the marker set was 2335 cM. Average markerheterozygosity was 0.57 and the mean intermarker spac-ing was 17 cM. The locations of markers are shown in Fig-ure 1 and 2.

Allele frequenciesA non-parametric linkage analysis using the softwareALLEGRO 1.0 [46], requires allele frequencies for markersif one parent is unknown and the transmission of allelesare not unambiguously traceable from the marker geno-types of the other parent and offspring. In our animalmaterial some of the dams and a few sires were missing.Marker allele frequencies in the population were esti-mated from our data set using a computer program madefor this purpose.

Linkage analysisTwo different test statistics were applied for linkage anal-ysis using the software package ALLEGRO 1.0 [46]. Thefirst statistic was numbers of shared alleles (NSA), i.e. thenumber of times that two affected full-sibs displayed iden-tical alleles at the two flanking markers. Under the nullhypothesis of no correlation between marker genotypesand defect status, two affected full-sibs would be expectedto share half their alleles. Under the alternative hypothesisof a linked gene affecting defect prevalence, the propor-tion of alleles being identical by descent (and accordingly,identical by state) would be expected to be higher. Thesecond test statistic considers how many of the sharedalleles of an affected sib-pair are identical by the descent(IBD). Multipoint linkage analysis of the genome scandata was performed with the non-parametric affected rel-ative method of ALLEGRO 1.0 [46], which also providesmultipoint NPL scores and the corresponding p-values[47].

Transmission/Disequilibrium testTransmission/Disequilibrium Tests [21] were carried outaccording to Lazzeroni and Lange [18]. At every locus, theTDT statistic was calculated as

where and are the number of times allele j has been

passed on from an heterozygous parent to an affected or

unaffected offspring, respectively, and are the

number of times allele j has not been passed on from anheterozygous parent (carrying allele j) to an affected orunaffected offspring, respectively, and l is the number ofalleles at the locus. The TDT test thus took both affectedand non-affected siblings into account. A permutation test

was carried out according to Lazzeroni and Lange [18], tocorrect for multiple loci and multiple alleles per locus. Foreach iteration of the permutation procedure, a permuteddata set was made by sampling, for each parent-offspringpair, a haplotype inherited by the offspring from that par-ent. The two possible, and equally likely, haplotypes werei) the haplotype inherited in the real data, and ii) the hap-lotype not inherited in the real data. In cases where inher-itance from parents to offspring could not be determinedin the real data set, because offspring and both parentswere heterozygous and identical, linkage phase in the off-spring was randomly assigned. TDT statistics were thencalculated as above, using the permuted data set. Follow-ing the last of m permutations, TDT statistics ti for all loci

i (from the real data set and all permutations) were con-verted to p-values using the empirical estimate;

where aki = 1 if Tki ≥ ti and aki = 0 otherwise (meaning thatpi(ti) is the rate at which the permuted test statistic waslarger than the true test statistic). Finally, adjusted p-val-ues (accounting for multiple loci) for the real data werecalculated as:

where bki = 1 if pi(ti) ≥ minm, with minm being the smallestp-value obtained across loci for permutation m. A VisualBasic program was written to perform this analysis, since,to our knowledge, the permutation option and the optionof using both affected and unaffected offspring is notavailable in any publicly available programs.

In addition to the locus-level TDT described above, TDTwas also performed on individual alleles, at single loci orhaplotypes. The TDT statistic (T) was calculated usingequation 1, and nominal significance levels were used, Tbeing distributed approximately as χ2 with one degree offreedom. For haplotype TDT, only those haplotypes fall-ing in the significant marker regions from the TDT or ASPtests were considered. Haplotypes were retrieved fromALLEGRO output.

Information contentThe information content (Info) supplied by ALLEGRO isdisplayed in Figure 1 and 2. The information contentranges from 0 to 1 (with 1 reflecting complete knowledgeof inheritance) and provides a measure of to what extendthe information present in the pedigrees could beextracted from the available genotypes, compared to a

Tt c t c

t c t c

ja

ju

ju

ja

ja

ju

ju

ja

j

l=

+ − +( )+ + +

[ ]=∑

1

1Eq.

t ja t j

u

c ja c j

u

p tm

ai i kik

m

( ) = [ ]=∑1 2

1

, .Eq

p p tm

bi i kik

m[ ] , .( ) = [ ]

=∑1 3

1

Eq



fully informative situation. It therefore allows an overviewof which chromosomes were sufficiently covered by mark-ers and in which chromosomal regions additional mark-ers might increase the power of the QTL studyconsiderably.

AbbreviationsASP, affected sib pair; LD, linkage disequilibrium; NPL,non-parametric linkage score; TDT, transmission/disequi-librium test; NSA, numbers of shared alleles; info, infor-mation content; INSL3, Insulin like receptor 3; MIS,Müllerian inhibiting substance; CGRP, Calcitonin gene-related peptide; ESR1, estrogen receptor 1; COL9A1, colla-gen typeIXα; COL2A1, collagen type II; INSL5, Insulin-like hormone 5; CYP19A1, cytochrome P450 family19A1;MAS, master assisted selection.

Authors' contributionsEG coordinated the study, organised and carried outmolecular genetic work, performed the statistical analysisof QTL mapping and drafted the paper. MM carried outmolecular genetic work. HT was involved in the statisticalanalysis. HS was involved in planning the project, powercalculations, supervising the statistical analysis and writ-ing the paper. SL was involved in planning the project andwriting the paper. TM performed the TDT statistical anal-ysis and contributed in writing the paper.

AcknowledgementsWe want to thank Emma Stubsjøen in Norsvin for a valuable contribution in organising the sample collection, and the technicians in Norsvin for taking blood samples. Thanks also to Arne Roseth and Brita Dalsgard for technical assistance in the lab. We are also very grateful to Heine Kristensen and Danish Slaughterhouses for providing us blood samples from hernia pigs in Denmark, to Olav Eik-Nes and Håvard Tajet for their contribution in initi-ating this project, and to Matthew Kent for valuable comments to the paper. NORSVIN (the Pig Breeders Association in Norway), the Research Council of Norway and the Swedish "Stiftelsen Lantbruksforskning" pro-vided financial support.

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AbstractBackgroundResultsConclusion

BackgroundResultsLinkage analysisTDT analysis

DiscussionConclusionMethodsAnimals and phenotypic recordsGenotypingStatistical analysisMarker mapAllele frequenciesLinkage analysisTransmission/Disequilibrium testInformation content

AbbreviationsAuthors' contributionsAcknowledgementsReferences

bmc genetics biomed central...nias are hernia inguinalis and hernia scrotalis which occur within the...

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